MOLECULAR AND RECOMBINANT TECHNOLOGY
Academic year and teacher
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- Versione italiana
- Academic year
- 2022/2023
- Teacher
- ALESSIA FINOTTI
- Credits
- 6
- Didactic period
- Secondo Semestre
- SSD
- BIO/11
Training objectives
- The course is aimed at teaching the techniques and the basic methodologies to study and analyze nucleic acids and gene expression, with special attention to recombinant DNA technology.
The practical laboratory exercise will give the possibility to the student to apply the acquired knowledge and will focus on the most used methods in the common research laboratories. At the same time students will learn the use of basic instruments (e.g. the use pipettes and other common laboratory instruments), and will be addressed on the experimental steps for the analysis of the expression of specific transcripts: from the RNA extraction, its spectrophotometric quantification and qualitative analysis by electrophoresis on agarose gel; the production of cDNA by reverse transcription and quantification of a specific mRNA (RT-PCR).
KNOWLEDGE AND UNDERSTANDING
The student will learn:
- correct use of scientific terms
- the techniques used to purify and quantify nucleic acids
- the techniques used for genomic and transcriptomic analyses
- the basic elements to build a recombinant vector
- the different types of recombinant vectors.
ABILITY TO APPLY KNOWLEDGE AND UNDERSTANDING
The student will know:
- the DNA and RNA extraction methods and for its analysis
- the approach for genomics and transcriptomics analysis
- which technique should be used for gene cloning experiments
- the suitable vectors used for recombinant protein production
- the vectors used to analyze and modify gene expression. Prerequisites
- Knowledge of Chemistry, Biology, Biochemistry and Molecular Biology.
Course programme
- The course includes 40 hours (5 CFU) of classroom didactic lecturing and 12 hours (1 CFU) of laboratory exercises.
Main issues:
FIRST SECTION: Methodologies for the analysis of nucleic acids and their expression and/or modulation.
- Extraction and purification of DNA, quantification and analysis: electrophoresis using agarose and Polyacrylamide gels, capillary electrophoresis;
- Techniques for genomic analysis: the employment of enzymes for DNA manipulation (e.g. restriction enzymes, polymerases, ligase), Southern blotting, Polymerase Chain Reaction and its variant, real-time qPCR, gene sequencing and Next Generation Sequencing;
- Extraction and purification of RNA and introduction to microRNAs;
-Assays for gene expression studies: reverse transcription, quantitative real-time PCR, digital PCR, transcriptomic analysis: gene expression arrays and RNAseq (these lessons will be held by Dr. Lucia Carmela Cosenza);
SECOND SECTION: Techniques of DNA modification, cloning vectors, expression vectors, transfection systems, and biotechnological applications.
- Enzymes for DNA cloning and basic principles of recombinant technologies;
- Molecular cloning vectors and their applications (plasmids, bacteriophages, cosmids, etc.);
- Transfection and other methods of introducing foreign DNA into living cells;
-Introduction to gene therapy vectors;
-Methods for recombinant protein production: vectors, expression systems for recombinant proteins, pharming, production of transgenic animals;
THIRD SECTION: Techniques for the study of gene expression and function.
-Gene and promoter identification;
-Assay for promoter and transcription factors analyses (e.g. EMSA and ChIP)
-Reporter gene assays;
-Study of gene methylation;
-In-vitro mutagenesis
-Use of RNA interference and gene silencing;
-Study of microRNAs Didactic methods
- The course is structured in 40 hours of classroom didactic lecturing.
During the didactic lecture, the course topics will be explained. In the classroom will be also uploaded useful scientific journals, websites, as references and explainer videos. The PowerPoint presentation of the topics will be provided by the teacher after the lesson (PDF format).
Suggested books are recommended for a more simple understanding of the basic concepts, but they do not talk about all the arguments explained by teaching and learning in the classroom.
In addition to the 40 hours of lectures (5CFU), it will be held a laboratory exercise (1CFU) divided in a section of practical laboratory experience and a section of preparation and analysis of the results carried out using PowerPoint lectures, data analysis and exercises with the possible assistance of videos and animations.
For the practical laboratory exercise, the students will be divided into groups and will work individually or in groups. Learning assessment procedures
- Molecular and Recombinant Technologies (Dr. Alessia Finotti):
The written exam includes 17 questions with multiple choice (with 5 answers only one of which is correct) and will be carried out with Google Forms modules. The exam will last up to 25 minutes.
For DSA students, the test procedure will be evaluated individually.
Each correct answer holds 2 points. To pass the exam (vote 18/30) it will be necessary to correctly answer at least 9 questions. The vote of 30/30 and honors will be achieved by answering correctly to 16 or 17 questions. No penalty points will be counted for wrong answers or unspecified answers. Reference texts
- Molecular and Recombinant Technologies (Dr. Alessia Finotti):
Slides were shown during the frontal lectures (PDF format); other material provided by the teacher.
Principal suggested text:
-Biotecnologie Molecolari. Principi e tecniche. Terry A. Brown - ZANICHELLI
Any further information on the following texts:
-Metodologie biochimiche e biomolecolari. M. Maccarrone - ZANICHELLI
-Genomi 4. IV edizione, 2018. Terry A. Brown - EDISES
-DNA ricombinante. Geni e Genomi. J.D. Watson et al. - ZANICHELLI
-Analisi dei Geni e Genomi. Richerd J. Reece - EdiSES
-Compendio di Biotecnologie Farmaceutiche. Maria Luisa Calabrò - EdiSES
-Metodologie Biochimiche. Bonaccorsi di Patti, Contestabile, Di Salvo - ZANICHELLI
